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Adhesion antigen SSUST3_1962 of streptococcus suis serotype 3, and preparation method and application thereof

A Streptococcus suis, adhesion technology, applied in the field of the adhesion antigen SSUST3_1962 of Streptococcus suis serotype 3 and its preparation, can solve the problem of not effectively controlling SS3

Inactive Publication Date: 2016-02-24
WUHAN TIANZHONG ANIMAL HUSBANDRY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no effective method and vaccine to control the infection of SS3, and there is an urgent need to develop a vaccine to effectively control the infection of SS3.

Method used

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  • Adhesion antigen SSUST3_1962 of streptococcus suis serotype 3, and preparation method and application thereof
  • Adhesion antigen SSUST3_1962 of streptococcus suis serotype 3, and preparation method and application thereof
  • Adhesion antigen SSUST3_1962 of streptococcus suis serotype 3, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Cloning, expression, purification and Western blot analysis of SSUST3_1962

[0042] 1) Using the genome of the SS3ST3 strain as a template to perform PCR to obtain a PCR product, wherein the PCR product contains the nucleotide sequence SEQIDNo.2;

[0043] Primer pairs (each primer contains a restriction site) are:

[0044] Forward primer: 5'-GTGGGTGGATCCACGGCGATTATCT-3';

[0045] Reverse primer: 5'-TCTTTTTTGAATTCAGGAGATGGCG-3'.

[0046] 2) The PCR product is digested with a suitable restriction enzyme and ligated with the pET43.1a vector;

[0047] 3) After transforming Escherichia coli, incubate at 37°C until the exponential growth phase;

[0048] 4) Add 1mMIPTG and incubate at 37°C for 3h;

[0049] 5) Collect the bacteria, add the loading buffer SDS-PAGE to run the gel;

[0050] 6) The recombinant protein was analyzed by western blot using the serum of recovered pigs infected with SS3.

[0051] Experimental result 1: The 86kDa recombinant protein was ex...

Embodiment 2

[0053] Example 2 Mice immunization and challenge test

[0054] 1) Thirty 4-week-old BALB / c female mice were randomly divided into 3 groups, 10 in each group;

[0055]2) 50 μg of purified recombinant SSUST3_1962 protein was emulsified with complete Freud adjuvant at a concentration of 50-100 μg / ml, and intraperitoneally injected into the mice of group 1;

[0056] 3) 14 days after the first immunization of the mice in group 1, inject the mice with 50 μg of the same antigen emulsified with incomplete adjuvant for the second time in the same way;

[0057] 4) Group 2 mice were injected with SEZ inactivated vaccine as a positive control (using complete or incomplete adjuvant emulsified inactivated SS3ST3 strain, the bacterial concentration was 2×10 8 CFU / ml);

[0058] 5) The mice in group 3 were injected with PBS emulsified with the same adjuvant as a negative control;

[0059] 6) 10 days after all mice were immunized, blood was obtained from the tail vein to obtain serum, and th...

Embodiment 3

[0063] Example 3 Antibody Titer Determination

[0064] Serum was determined by ELISA for IgG titer, and the microtiter plate was coated with purified recombinant protein SSUST3_1962. Serum was obtained from the booster immunized mice, and after the serum was serially diluted (100 times for the first dilution), IgG and IgG isotype antigens were used to detect the GAPDH antibody titer. The antibody titer is the reciprocal of the 50% maximum OD value of the serum dilution. In order to infer the immune type, IgG1 and IgG2a were further used as controls to measure Th2 and Th1 immune responses, respectively.

[0065] Experimental result 1: The IgG titer GAPDH antibody level of the immune group was significantly higher than that of the negative control group (p figure 2 A).

[0066] Experimental result 2: We did not accurately determine the type of immunoglobulin, but the results did not show that SSUST3_1962 can induce a significant Th1- / Th2 immune response, and IgG1 and IgG2 resp...

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Abstract

The invention discloses an adhesion antigen SSUST3_1962 of streptococcus suis serotype 3 (SS3), and a preparation method and application thereof. According to the invention, SSUST3_1962 recombinant protein is constructed, and the protein can adhere to Hep-2 cells so as to interfere in infection of cells with SS3. In the process of research in the invention, a purified recombinant antigen can cause substantial humoral antibody response of a mouse, and an immunized mouse shows strong protection capability to challenge of a lethal dose of SS3. Moreover, whole blood analysis test results show that secondarily immunized serum of SSUST3_1962 can effectively inhibit growth of bacteria. All the research results show that the SSUST3_1962 recombinant protein can be used for preparing a subunit vaccine for preventing and treating SS3 infection.

Description

technical field [0001] The invention relates to the field of antigen and antibody of pigs; in particular, it refers to an adhesion antigen SSUST3_1962 of Streptococcus suis serotype 3 and its preparation method and application. Background technique [0002] Streptococcus suis (SS) causes a range of symptoms in pigs, including meningitis, septicemia, polyserositis, arthritis, endocarditis and pneumonia. So far, Streptococcus suis is divided into 33 serotypes, and the pathogenicity of different serotypes and different strains of the same type is different. The most prevalent in my country's pig herds is serotype 2 Streptococcus suis, followed by serotype 3 Streptococcus suis. Due to the lack of comprehensive understanding of SS3 virulence factors and protective antigens, it is difficult to effectively control SS3 infection. Therefore, preventing the spread of this pathogen relies on strict control measures. [0003] At present, there is no effective method and vaccine for c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/315C12N15/31C12N15/70C12N1/21A61K39/09A61P31/04
CPCA61K39/00C07K14/315
Inventor 章红军赵祖凯魏子贡樊翠华罗小锋
Owner WUHAN TIANZHONG ANIMAL HUSBANDRY CO LTD
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